In vitro manipulation of pollen for transformation in cotton and Tomato
KrishiKosh
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Title |
In vitro manipulation of pollen for transformation in cotton and Tomato
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Creator |
D.Satish
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Contributor |
R.L.Ravikumar
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Subject |
Agronomy
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Description |
The laboratory and field experiments on pollen transformation in cotton and tomato were carried out during 2004-20007 at the Department of GPB, UAS, Dharwad. The main objective of the study was to assess the potentiality of production of transgenic cotton and tomato plants consistently using pollen as vector. In the present study, in vitro pollen germination medium for cotton variety Sahana and tomato variety Pusa ruby were standardized. The co-cultivation of pollen with Agrobacterium tumefaciens strain LBA4404 containing plasmid vector pCAMBIA1305.1 significantly reduced the germination per cent and tube growth compared to control both in cotton and tomato. The co-cultivation of cotton pollen with Agrobacterium tumefaciens containing GUS as a reporter gene in cavity slides resulted in 3.11 percentage of pollen grains showing transient GUS expression. The co-cultivated pollen grains were used for pollination. To favour the transformed pollen grains in fertilization, the style and stigma of the emasculated flowers were treated with various concentrations of hygromycin (250, 500 and 1000 ppm in cotton and 250,500,1000 and 1500 ppm in tomato). The increased concentration of hygromycin to stigma and style has significantly reduced the boll set in cotton and fruit set in tomato. The number of seeds obtained from each treatment also varied. Totally, 223 cotton plants and 390 tomato plants were screened for presence and expression of transgene. In cotton, 32 plants (14.34%) showed resistance to hygromycin application, 14 plants (6.27%) were positive for PCR amplification and 12 plants (5.38%) were positive for GUS assay in leaves in T0 generation. In case of tomato, 13 plants (3.3%) were resistant to hygromycin application. Only two plants (0.15%) were positive for PCR amplification and one plant (0.25%) showed positive for GUS staining in leaves in T0 generation. Further, the positive plants were advanced to T1 generation. The T1 plants showed resistant to hygromycin application and positive for PCR amplification, suggesting stable integration of DNA introduced through pollen grains. In order to increase the frequency of pollen grains with transgene, different pretreatments to pollen grains were given. The pre- hydration of pollen for 30 minutes, pretreating cotton pollen with 40 and 45% PEG and tomato pollen with 12% PEG for 2 hours, pre germination of pollen grains for 45 minutes. The pre-treatments increased the frequency of transgene insert into the pollen significantly. Further, parameters of particle bombardment were optimized for transformation of cotton pollen grains. Two particle size (0.7& 1.1μ), two shooting distance (6 cm &9 cm) and three pollen treatments (pre- hydration, PEG treatment and control) were used. The bombardment of PEG pre- treated pollen grains with 1.1μ particles at a target distance of 9 cm produced the highest frequency (29.04%) of transient GUS expressing pollens. |
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Date |
2016-07-23T09:08:09Z
2016-07-23T09:08:09Z 2009 |
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Type |
Thesis
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Identifier |
http://krishikosh.egranth.ac.in/handle/1/69451
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Format |
application/pdf
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Publisher |
UAS Dharwad
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